The pharmacological efficacy of a protein often depends on the protein's glycosylation structure, and successful manufacturing relies on host systems that produce glycoforms similar to those in humans. The overall objectives of this project are to develop tools for the metabolic engineering of fungal glycosylation pathways and to use these tools toward the creation of fungal strains capable of producing and secreting large quantities of recombinant therapeutic proteins with human-like glycosylation structures. The tool development strategy centers on a library-based approach that will allow rapid identification of properly targeted and expressed glycosylation enzymes in a high throughput manner. PROPOSED COMMERICIAL APPLICATIONS: The current method of glycoprotein production based on mammalian cell culture systems, specifically Chinese hamster ovary cell lines and mouse fibroblast/myeloma cell lines, presents manufacturers with the problems of low production levels, high cost, and safety. In its present state, protein production technology cannot keep up with research and development efforts, making manufacturing the growth-limiting factor for the biotechnology industry. With 84 biological drugs on the market and well over 500 in clinical development, the proposed project will generate technology toward the manufacturing of glycosylated proteins with high production levels in a safe and cost-effective manner, thereby alleviating the production bottleneck and allowing more products to reach the patients who need them.